Azo compounds for type I phototherapy

ABSTRACT

Novel azo compounds and their bioconjugates for phototherapy and/or photodiagnosis of tumors and other lesions. The azo derivatives of the present invention are designed to absorb at the low-energy ultraviolet, visible, or near-infrared (NIR) region of the electromagnetic spectrum. The phototherapeutic effect is caused by direct interaction of free radicals, the reactive intermediate produced upon photoexcitation of the azo compound, with the tissue of interest.

This application is a continuation-in-part of U.S. application Ser. No.09/849,163 filed May 4, 2001, now U.S. Pat. No. 6,485,704 and expresslyincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention relates generally to novel azo bioconjugates for use inphototherapy.

BACKGROUND OF THE INVENTION

The use of visible and near-infrared (NIR) light in clinical practice isgrowing rapidly. Compounds absorbing or emitting in the visible, NIR, orlong-wavelength (UV-A, >350 nm) region of the electromagnetic spectrumare potentially useful for optical tomographic imaging, endoscopicvisualization, and phototherapy. However, a major advantage ofbiomedical optics lies in its therapeutic potential. Phototherapy hasbeen demonstrated to be a safe and effective procedure for the treatmentof various surface lesions, both external and internal. Its efficacy iscomparable to that of radiotherapy, but without the harmfulradiotoxicity of critical non-target organs.

Phototherapy has been in existence for many centuries and has been usedto treat various skin surface ailments. As early as 1400 B.C. in India,plant extracts (psoralens), in combination with sunlight, were used totreat vitiligo. In 1903, Von Tappeiner and Jesionek used eosin as aphotosensitizer for the treatment of skin cancer, lupus of the skin, andcondylomata of female genitalia. Over the years, the combination ofpsoralens and ultraviolet A (low-energy) radiation has been used totreat a wide variety of dermatological diseases including psoriasis,parapsoriasis, cutaneous T-cell lymphoma, eczema, vitiligo, areata, andneonatal bilirubinemia. Although the potential of cancer phototherapyhas been recognized since early 1900's, systematic studies todemonstrate safety and efficacy began only in 1967 with the treatment ofbreast carcinoma. Dougherty et al. subsequently conclusively establishedthat long-term cure is possible with photodynamic therapy (PDT).Currently, phototherapeutic methods are also being investigated for thetreatment of some cardiovascular disorders such as atherosclerosis andvascular restenosis for the treatment rheumatoid arthritis, and for thetreatment of some inflammatory diseases such as Crohn's disease.

Phototherapeutic procedures require photosensitizers (i.e. chromophores)which have high absorptivity. These compounds should preferably bechemically inert, and become activated only upon irradiation with lightof an appropriate wavelength. Light-initiated selective tissue injurycan be induced when these photosensitizers bind to target tissues,either directly or through attachment to a bioactive carrier.Furthermore, if the photosensitizer is also a chemotherapeutic agent(e.g. anthracycline antitumor agents), then an enhanced therapeuticeffect can be attained.

Effective phototherapeutic agents should have the following properties:(a) large molar extinction coefficient; (b) long triplet lifetime; (c)high yield of singlet oxygen and/or other reactive intermediates, viz.,free radicals, nitrenes, carbenes, open-shell ionic species such ascabonium ions and the like; (d) efficient energy or electron transfer tocellular components; (e) low tendency to form aggregation in aqueousmilieu; (f) efficient and selective targeting of lesions; (g) rapidclearance from blood and non-target tissues; (h) low systemic toxicity;and (i) lack of mutagenicity.

Photosensitizers operate via two distinct pathways, termed Types 1 and2. The type 1 mechanism is shown in the following scheme:

After photoexcitation, the Type 1 mechanism involves direct energy orelectron transfer from the photosensitizer to the cellular components,thereby causing cell death. After photoexcitation, the Type 2 mechanisminvolves distinct steps as shown in the following scheme:

In the first step, singlet oxygen is generated by energy transfer fromthe triplet excited state of the photosensitizer to the oxygen moleculessurrounding the tissues. In the second step, collision of a singletoxygen with the tissues promotes tissue damage. In both Type 1 and Type2 mechanisms, the photoreaction proceeds via the lowest triplet state ofthe sensitizer. Hence, a relatively long triplet lifetime is requiredfor effective phototherapy. In contrast, a relatively short tripletlifetime is required to avoid photodamage to the tissue caused byphotosensitizers.

The biological basis of tissue injury brought about by tumorphototherapeutic agents has been the subject of intensive study. Variousreasonable biochemical mechanisms for tissue damage have been postulatedeven though the type and number of photosensitizers employed in thesestudies are relatively small. These biochemical mechanisms are asfollows: a) cancer cells upregulate the expression of low densitylipoprotein (LDL) receptors, and PDT agents bind to LDL and albuminselectively; (b) porphyrin-like substances are selectively taken up byproliferative neovasculature; (c) tumors often contain an increasednumber of lipid bodies and are thus able to bind to hydrophobicphotosensitizers; (d) a combination of “leaky” tumor vasculature andreduced lymphatic drainage causes porphyrin accumulation; (e) tumorcells may have increased capabilities for phagocytosis or pinocytosis ofporphyrin aggregates; (f) tumor associated macrophages may be largelyresponsible for the concentration of photosensitizers in tumors; and (g)cancer cells may undergo apoptosis induced by photosensitizers. Amongthese mechanisms, (f) and (g) are the most general and, of these twoalternatives, there is a general consensus that (f) is the most likelymechanism by which the phototherapeutic effect of porphyrin-likecompounds is induced.

Most of the currently known photosensitizers are commonly referred to asPDT agents and operate via the Type 2 mechanism. For example, PhotofrinII, a hematoporphyrin derivative, was approved by the United States Foodand Drug Administration for the treatment of bladder, esophageal, andlate-stage lung cancers. However, Photofrin II has been shown to haveseveral drawbacks: low molar absorptivity, (ε=3000M⁻¹), low singletoxygen quantum yield (φ=0.1), chemical heterogeneity, aggregation, andprolonged cutaneous photosensitivity. Hence, there has been considerableeffort in developing safer and more effective photosensitizers for PDTthat exhibit improved light absorbance properties, better clearance, anddecreased skin photosensitivity compared to those of Photofrin II. Thesephotosensitizers include monomeric porphyrin derivatives, corrins,cyanines, phthalocyanines, phenothiazines, rhodamines, hypocrellins, andthe like. However, these phototherapeutic agents also mainly operate viathe Type 2 mechanism.

Surprisingly, there has not been much attention directed at developingType 1 phototherapeutic agents, despite the fact that the Type 1mechanism seems inherently more efficient than the Type 2 mechanism.First, unlike Type 2, Type 1 photosensitizers do not require oxygen forcausing cellular injury. Second, the Type 1 mechanism involves two steps(photoexcitation and direct energy transfer) whereas the Type 2mechanism involves three steps (photoexcitation, singlet oxygengeneration, and energy transfer). Furthermore, some tumors have hypoxicregions that render the Type 2 mechanism ineffective. In spite of thedrawbacks associated with the Type 2 mechanism, however, only a smallnumber of compounds have been developed that operate through the Type 1mechanism, e.g. anthracyline antitumor agents.

Thus, there is a need to develop effective phototherapeutic agents thatoperate through the Type 1 mechanism. Phototherapeutic efficacy can befurther enhanced if the excited state photosensitizers can generatereactive intermediates such as free radicals, nitrenes, carbenes, andthe like. These have much longer lifetimes than the excited chromophoreand have been shown to cause considerable cell injury.

SUMMARY OF THE INVENTION

The present invention addresses this need and discloses novel azoderivatives and their bioconjugates that absorb in the low-energy,ultraviolet, visible, or near-infrared (NIR) region of theelectromagnetic spectrum that are used for the phototherapy of tumorsand other lesions. More specifically, the present invention disclosesazo compounds having the formula 1

where Q is a single bond or —CR¹R²; R¹ and R² are independently selectedfrom the group consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, C1-C10alkoxyalkyl; C1-C10 polyhydroxyalkyl, —(CH₂)_(a)CO₂H, and—(CH₂)_(b)NR³R⁴; R³ and R⁴ are independently selected from the groupconsisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, C1-C10polyhydroxyalkyl, and —(CH₂)_(a)CO₂H; R⁵, R⁶, and R⁷ are independentlyselected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10aryl, hydroxyl, —SO₃H, C1-C10 alkoxyl, C1-C10 polyhydroxyalkyl, C1-C10polyalkoxyalkyl, —(CH₂)_(a)CO₂H, and —(CH₂)_(b)NR³R⁴; X is selected fromthe group consisting of —CR⁸R⁹, —O—, —NR³, —S—, and —C═O; Y is selectedfrom the group consisting of —CR¹⁰R¹¹, —O—, —NR³, —S—, and —C═O; Z isselected from the group consisting of —CR¹²R¹³, —O—, —NR³, —S—, and—C═O; R⁸ to R¹³ are independently selected from the group consisting ofhydrogen, C1-C10 alkyl, C5-C10 aryl, C1-C10 alkoxyalkyl, C1-C10polyhydroxyalkyl, —(CH₂)_(a)CO₂H, and —(CH₂)_(b)NR³R⁴; R⁵-R⁶, R⁶-R⁷,R⁸-R¹⁰, or R¹⁰-R¹² together optionally form a six-membered alicyclic oraromatic ring; E is either a hydrogen atom or is selected from the groupconsisting of antibodies, peptides, peptidomimetics, carbohydrates,glycomimetics, drugs, hormones, or nucleic acids; L is a linker unitselected from the group consisting of —(CH₂)_(c)—, —(CH₂)_(d)CONR³—,—N(R³)CO(CH₂)_(d)—, —OCO(CH₂)_(e)—, —(CH₂)_(f)CO₂—, —OCONH—, —OCO₂—,—HNCONH—, —HNCSNH—, —HNNHCO—, —OSO₂—, —NR³(CH₂)_(g)CONR⁴—,—CONR³(CH₂)_(h)NR⁴CO—, and —NR³CO(CH₂)_(i)CONR⁴; a, b, d to iindependently range from 0 to 10, and c ranges from 1 to 10.

The present invention also discloses a method of performing aphototherapeutic or photodiagnostic procedure using the inventive azocompounds and their derivatives. In the method, an effective amount ofan azo photosensitizer having the formula 1

is administered to a subject; where Q is a single bond or —CR¹R²; R¹ andR² are independently selected from the group consisting of hydrogen,C1-C10 alkyl, C5-C10 aryl, C1-C10 alkoxyalkyl, C1-C10 polyhydroxyalkyl,—(CH₂)_(a)CO₂H, and —(CH₂)_(b)NR³R⁴; R³ and R⁴ are independentlyselected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10aryl, C1-C10 polyhydroxyalkyl, and —(CH₂)_(a)CO₂H; R⁵, R⁶, and R⁷ areindependently selected from the group consisting of hydrogen, C1-C10alkyl, C5-C10 aryl, hydroxyl, —SO₃H, C1-C10 alkoxyl, C1-C10polyhydroxyalkyl, C1-C10 polyalkoxyalkyl, —(CH₂)_(a)CO₂H, and—(CH₂)_(b)NR³R⁴; X is selected from the group consisting of —CR⁸R⁹, —O—,—NR³, —S—, and —C═O; Y is selected from the group consisting of—CR¹⁰R¹¹, —O—, —NR³, —S—, and —C═O; Z is selected from the groupconsisting of —CR¹²R¹³, —O—, —NR³, —S—, and —C═O; R⁸ to R¹³ areindependently selected from the group consisting of hydrogen, C1-C10alkyl, C5-C10 aryl, C1-C10 alkoxyalkyl, C1-C10 polyhydroxyalkyl,—(CH₂)_(a)CO₂H, and —(CH₂)_(b)NR³R⁴; R⁵-R⁶, R⁶-R⁷, R⁸-R¹⁰, or R¹⁰-R¹²together optionally form a six-membered ring; E is either a hydrogenatom or is selected from the group consisting of antibodies, peptides,peptidomimetics, carbohydrates, glycomimetics, drugs, hormones, ornucleic acids; L is a linker unit selected from the group consisting of—(CH₂)_(c)—, —(CH₂)_(d)CONR³—, —N(R³)CO(CH₂)_(d)—, —OCO(CH₂)_(e)—,(CH₂)_(f)CO₂—, —OCONH—, —OCO₂—, —HNCONH—, —HNCSNH—, —HNNHCO—, —OSO₂—,—NR³(CH₂)_(g)CONR⁴—, —CONR³(CH₂)_(h)NR⁴CO—, and —NR³CO(CH₂)_(i)CONR⁴; a,b, d to i independently range from 0 to 10, and c ranges from 1 to 10.The compound is photoactivated and a phototherapeutic or photodiagnosticprocedure for tumors, impaired vasculature or other lesions issubsequently performed.

For targeting purposes, external attachment of an epitope is used unlessthe azo compounds themselves preferentially accumulate in the targettissue. For example, if the photosensitizing chromophore is ananthracycline moiety, it can bind to cancer cells directly and may notrequire an epitope for targeting purposes.

These and other advantages and embodiments of the inventive compoundsand methods will be apparent in view of the following figures,description, and example.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic pathway for activation of the inventive compounds.

FIG. 2 is a schematic pathway for the synthesis of a cyclic azoxanthenederivative.

FIG. 3 is a schematic pathway for the synthesis of an azoacridinederivative.

FIG. 4 is a schematic pathway for the synthesis of an azocoumarinderivative.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses novel azo derivatives and theirbioconjugates for phototherapy of tumors and other lesions.

Accordingly, the present invention provides new and structurally diversecompositions comprising organic azo compounds of the general formula 1

wherein Q is a single bond or —CR¹R²; R¹ and R² are independentlyselected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10aryl, C1-C10 alkoxyalkyl, C1-C10 polyhydroxyalkyl, —(CH₂)_(a)CO₂H, and—(CH₂)_(b)NR³R⁴; R³ and R⁴ are independently selected from the groupconsisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, C1-C10polyhydroxyalkyl, and —(CH₂)_(a)CO₂H; R⁵, R⁶, and R⁷ are independentlyselected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10aryl, hydroxyl, —SO₃H, C1-C10 alkoxyl, C1-C10 polyhydroxyalkyl, C1-C10polyalkoxyalkyl, —(CH₂)_(a)CO₂H, and —(CH₂)_(b)NR³R⁴; X is selected fromthe group consisting of —CR⁸R⁹, —O—, —NR³, —S—, and —C═O; Y is selectedfrom the group consisting of —CR¹⁰R¹¹, —O—, —NR³, —S—, and —C═O; Z isselected from the group consisting of —CR¹²R¹³, —O—, —NR³, —S—, and—C═O; R⁸ to R¹³ are independently selected from the group consisting ofhydrogen, C1-C10 alkyl, C5-C10 aryl, C1-C10 alkoxyalkyl, C1-C10polyhydroxyalkyl, —(CH₂)_(a)CO₂H, and —(CH₂)_(b)NR³R⁴; R⁵-R⁶, R⁶-R⁷,R⁸-R¹⁰, or R¹⁰-R¹² together optionally form a six-membered ring; E iseither a hydrogen atom or is selected from the group comprisingantibodies, peptides, peptidomimetics, carbohydrates, glycomimetics,drugs, hormones, or nucleic acids; L is a linker unit selected from thegroup comprising —(CH₂)_(c)—, —(CH₂)_(d)CONR³—, —N(R³)CO(CH₂)_(d)—,—OCO(CH₂)_(e)—, —(CH₂)_(f)CO₂—, —OCONH—, —OCO₂—, —HNCONH—, —HNCSNH—,—HNNHCO—, —OSO₂—, —NR³(CH₂)₂CONR⁴—, —CONR³(CH₂)_(h)NR⁴CO—, and—NR³CO(CH₂)_(i)CONR⁴; a,b, d to i independently range from 0 to 10, andc ranges from 1 to 10.

In one embodiment, azo compounds according to the present invention havethe general formula 1 wherein Q is —CR¹R²; R¹ and R² are independentlyselected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10aryl, and —(CH₂)_(a)CO₂H; R⁵, R⁶, and R⁷ are independently selected fromthe group consisting of hydrogen, C1-C10 alkyl, hydroxyl, —SO₃H, C1-C10alkoxyl, and —(CH₂)_(a)CO₂H; X is selected from the group consisting of—CR⁸R⁹, —O—, —NR³, and —C═O; Y is selected from the group consisting of—CR¹⁰R¹¹, —O—, —NR³, and —C═O; Z is selected from the group consistingof —CR¹²R¹³, —O—, —NR³, and —C═O; R⁸ to R¹³ are independently selectedfrom the group consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, and—(CH₂)_(a)CO₂H; R⁸ and R¹⁰ together optionally form a six-membered ring;E is selected from the group consisting of somatostatin receptor bindingmolecules, heat-sensitive bacterioendotoxin (ST) receptor bindingmolecules, neurotensin receptor binding molecules, bombesin receptorbinding molecules, cholecystekinen (CCK) receptor binding molecule,steroid receptor binding molecules, and carbohydrate receptor bindingmolecules; L is a linker unit selected from the group consisting of—(CH₂)_(d)CONR³—, —N(R³)CO(CH₂)_(d)—, —HNCONH—, —HNCSNH—, and—NR³CO(CH₂)_(i)CONR⁴; and a to i independently range from 0 to 6.

In another embodiment, azo compounds according to the invention havingthe general formula 1 above wherein Q is —CR¹R²; R¹ and R² areindependently selected from the group consisting of hydrogen and C1-C10alkyl; R⁵, R⁶, and R⁷ are independently selected from the groupconsisting of hydrogen, hydroxyl, —SO₃H, and —(CH₂)_(a)CO₂H; X isselected from the group consisting of —CR⁸R⁹ and —C═O; Y is selectedfrom the group consisting of —CR¹⁰R¹¹, —NR³, and —C═O; Z is selectedfrom the group consisting of —CR¹²R¹³, —O—, —NR³, and —C═O; R⁸ to R¹³are independently selected from the group consisting of hydrogen, C1-C10alkyl, C5-C10 aryl, and —(CH₂)_(a)CO₂H; R⁸ and R¹⁰ together optionallyform a six-membered ring; E is selected from the group consisting ofsomatostatin receptor binding molecules, ST receptor binding molecules,neurotensin receptor binding molecules, bombesin receptor bindingmolecules, CCK receptor binding molecule, steroid receptor bindingmolecules, and carbohydrate receptor binding molecules; L is a linkerunit selected from the group consisting of —N(R³)CO(CH₂)_(d)—,—(CH₂)_(d)CONR³—, and NR³CO(CH₂)_(i)CONR⁴; and a, b, d, e, f, g, h and iindependently range from 0 to 6.

In an additional embodiment, azo compounds according to the inventionhaving the general formula 1 wherein Q is a single bond or —CR¹R²; R¹and R² are independently selected from the group consisting of hydrogen,C1-C10 alkyl, C5-C10 aryl, C1-C10 alkoxyalkyl, C1-C10 polyhydroxyalkyl,—(CH₂)_(a)CO₂H, and —(CH₂)_(b)NR³R⁴; R³ and R⁴ are independentlyselected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10aryl, C1-C10 polyhydroxyalkyl, and —(CH₂)_(a)CO₂H; R⁵, R⁶, and R⁷ areindependently selected from the group consisting of hydrogen, C1-C10alkyl, C5-C10 aryl, hydroxyl, —SO₃H, C1-C10 alkoxyl, C1-C10polyhydroxyalkyl, C1-C10 polyalkoxyalkyl, —(CH₂)_(a)CO₂H, and—(CH₂)_(b)NR³R⁴; X is selected from the group consisting of —CR₈R⁹, —O—,—NR³, —S—, and —C═O; Y is selected from the group consisting of—CR¹⁰R¹¹, —O—, —NR³, —S—, and —C═O; Z is selected from the groupconsisting of —CR¹²R¹³, —O—, —NR³, —S—, and —C═O; R⁸ to R¹³ areindependently selected from the group consisting of hydrogen, C1-C10alkyl, C5-C10 aryl, C1-C10 alkoxyalkyl, C1-C10 polyhydroxyalkyl,—(CH₂)_(a)CO₂H, and —(CH₂)_(b)NR³R⁴; R⁵-R⁶, R⁶-R⁷, R⁸-R¹⁰, or R¹⁰-R¹²together optionally form a six-membered ring; E is either a hydrogenatom or is selected from the group comprising antibodies, peptides,peptidomimetics, carbohydrates, glycomimetics, drugs, hormones, ornucleic acids; L is the linker unit —(CH₂)_(c)—, a and b independentlyrange from 0 to 10, and c ranges from 1 to 10.

The inventive compounds operate through the Type 1 mechanism as shown inFIG. 1 wherein —N═N— is the azo moiety that undergoes nitrogen extrusionupon photoactivation, thereby producing free radicals. Ar is an aromaticchromophore that undergoes photosensitization. Aliphatic azo compoundscan also be used for phototherapy, but may require high-energy light foractivation. L is the linker between the chromophore and the epitope.Epitope (E) is a particular region of the molecule that is recognized byand binds to the target surface. An epitope is usually, but not always,associated with biomolecules. Biomolecules include hormones, aminoacids, peptides, peptidomimetics, proteins, nucleosides, nucleotides,nucleic acids, enzymes, carbohydrates, glycomimetics, lipids, albumins,mono- and polyclonal antibodies, receptors, inclusion compounds such ascyclodextrins, and receptor binding molecules. Specific examples ofbiomolecules include steroid hormones for the treatment of breast andprostate lesions; somatostatin, bombesin, CCK, and neurotensin receptorbinding molecules for the treatment of neuroendocrine tumors; CCKreceptor binding molecules for the treatment of lung cancer; ST receptorand carcinoembryonic antigen (CEA) binding molecules for the treatmentof colorectal cancer; dihyroxyindolecarboxylic acid and other melaninproducing biosynthetic intermediates for the treatment of melanoma;integrin receptor and atherosclerotic plaque binding molecules for thetreatment of vascular diseases; and amyloid plaque binding molecules forthe treatment of brain lesions. Examples of synthetic polymers includepolyaminoacids, polyols, polyamines, polyacids, oligonucleotides,aborols, dendrimers, and aptamers.

Coupling of a photodiagnostic and/or phototherapeutic agent tobiomolecules can be accomplished by methods well known in the art, asdisclosed in Hnatowich et al., Radiolabeling of Antibodies: A simple andefficient method, Science, 1983, 220, p. 613; Pelegrin et al.,Photoimmunodiagnostics with antibody-fluorescein conjugates: in vitroand in vivo preclinical studies, Journal of Cellular Pharmacology, 1992,3, pp. 141-145; and U.S. Pat. No. 5,714,342, each of which is expresslyincorporated by reference herein in its entirety. Successful specifictargeting of fluorescent dyes to tumors using antibodies and peptidesfor diagnostic imaging of tumors has been demonstrated by us and othersas described in Achilefu et al., Novel receptor-targeted fluorescentcontrast agents for in vivo imaging of tumors, Investigative Radiology,2000, 35, pp. 479-485; Ballou et al., Tumor labeling in vivo usingcyanine conjugated monoclonol antibodies, Cancer Immunology andImmunotherapy, 1995, 41, pp. 257-263; and Licha et al., New contrastagent for optical imaging: acid cleavable conjugates of cyanine dyeswith biomolecules, in Biomedical Imaging: Reporters, Dyes andInstrumentation. Proceedings of SPIE, 1999, 3600, pp. 29-35, each ofwhich is expressly incorporated by reference herein in its entirety.Therefore, receptor-targeted phototherapeutic agents of the presentinvention should be effective in the treatment of various lesions.

In the process outlined in FIG. 1, photoexcitation of the aromaticchromophore effects rapid intramolecular energy transfer to the azogroup, resulting in N—C bond rupture with concomitant extrusion ofmolecular nitrogen and formation of diradicals. The diradicals can alsocombine with each other to form neutral molecules, provided that theirspatial orientation is optimal. The nitrogen that is released could bein a vibrationally excited state and may cause additional cellularinjury. This process is very similar to the process observed withazides. For targeting purposes, an external attachment of an epitope isusually required unless the azo compounds themselves preferentiallyaccumulate in the target tissue, thereby obviating the need for anadditional binding group. For example, if the Ar moiety is ananthracycline moiety, it can bind to cancer cells directly and may notrequire an epitope for targeting purposes.

The synthesis of azo compounds is accomplished by a variety of methodswell known in the art, as disclosed in Sandier and Karo, Azo Compounds,Organic Functional Group Preparations, 1986, Academic Press: New York,pp. 353-409, which is expressly incorporated by reference herein in itsentirety. The azo derivatives of the invention contain additionalfunctionalities that can be used to attach various types ofbiomolecules, synthetic polymers, and organized aggregates for selectivedelivery to various organs or tissues of interest. Preparations ofrepresentative compounds from the embodiments are shown in FIGS. 2-4.

FIG. 2 shows a typical preparation of a cyclic azoxanthene derivative 5.Methyl 2-chloro-5-nitrobenzoate 1 is reacted with 3-hydroxybenzylalcohol 2 and thereafter saponified and cyclized to the nitroxanthone 3.The xanthone 3 is then converted to the azo precursor 4 in four standardsteps. The hydrazino derivative 4 is then oxidized with either mercuricoxide or lead tetraacetate and then conjugated to any desiredbiomolecule of interest using bifunctional coupling reagents such asphosgene, thiophosgene, carbonyldiimidazole, disuccinimidyl carbonate,and the like. Specifically, the biomolecule of the invention pertains tothose binding to colorectal, cervical, ovarian, lung, and neuroendocrinetumors. These include somatostatin, cholesystekinin (CCK), bombesin,neuroendrocrine, and heat sensitive bacterioendotoxin (ST) receptorbinding compounds.

With reference to FIG. 3, the azoacridine derivative 9 can be preparedin a similar manner to the cyclic azoxanthene derivative whose syntheticscheme is shown in FIG. 2. Methyl 2-chloro-5-nitrobenzoate 1 is reactedwith 3-hydroxybenzyl amine 6 and thereafter saponified and cyclized tothe nitroacridone 7. The acridone 7 is then converted to the azoprecursor 8 in four standard steps. The hydrazino derivative 8 is thenoxidized with either mercuric oxide or lead tetraacetate and thenconjugated to a biomolecule, as previously described, using bifunctionalcoupling reagents such as disuccinimidyl carbonate, disuccinimidyloxalate, phosgene, thiophosgene, carbonyidiimidazole and the like.

With reference to FIG. 4, a typical preparation of an azocoumarinderivative 12 is shown. The phenol 10 is first alkylated with methylbromoacetate and then transformed to the azo compound 11 by standardmethods. The ester 11 is saponified and conjugated to the biomoleculeusing the known bifunctional coupling reagents previously described, orcan be conjugated directly using automated peptide synthesis methods asis known to one of skill in the art.

The novel compositions of the present invention may vary widelydepending on the contemplated application. For tumors, the biomoleculeis selected from the class of tumor markers including, but not limitedto, somatostatin, bombesin, neurotensin, CCK, ST, estrogen, andprogesterone receptor binding compounds. For vascular lesions, thebiomolecule may be selected from the class of integrins, selecting,vascular endothelial growth factor, fibrins, tissue plasminogenactivator, thrombin, low density lipoprotein (LDL), high densitylipoprotein (HDL), Sialyl Lewis^(x) and its mimics, and atheroscleroticplaque binding compounds.

As previously described, some compounds accumulate in tumors or otherlesions without the assistance of a bioactive carrier. Administration ofdelta-aminolevulinic acid, an intermediate in porphyrin biosynthesis,results in a two-fold uptake of porphyrins in tumors compared to normaltissues. Similarly, administration of dihydroxyindole-2-carboxylic acid,an intermediate in melanin biosynthesis, produces substantially enhancedlevels of melanin in melanoma cells compared to normal cells. Thus, aphotosensitizer may be delivered to the site of lesion by attaching itto these types of biosynthetic intermediates.

Methods of performing therapeutic procedures with compositions of theinvention are also disclosed. The method encompasses administering to apatient an effective amount of the compositions of the inventioncontained in a pharmaceutically acceptable formulation. Thereafter, thephotosensitizer is allowed to accumulate in the region of interest,followed by illumination with light of wavelength 300 to 1200 nm,preferably 350 to 850 nm, at the site of the lesion. If the lesion is onthe skin surface, it can be directly illuminated; otherwise, endoscopiccatheters equipped with a light source may be employed to achieve aphototherapeutic effect. The intensity, power, duration of illumination,and the wavelength of the light may vary widely depending on thelocation and site of the lesions. The fluence rate is preferably, butnot always, kept below 200 mW/cm² to minimize thermal effects.Appropriate power depends on the size, depth, and pathology of thelesion. The inventive compositions have broad clinical utility whichincludes, but is not limited to, phototherapy of tumors, inflammatoryprocesses, and impaired vasculature.

The inventive compositions can be formulated into photodiagnostic orphototherapeutic compositions for enteral (oral or rectal), parenteral,topical, or cutaneous administration. Topical or cutaneous delivery ofthe photosensitizer may also include aerosols, creams, gels, solutions,etc. The compositions are administered in doses effective to achieve thedesired diagnostic or therapeutic objective. Such doses may vary widelydepending upon the particular complex employed, the organs or tissues tobe examined, the equipment employed in the clinical procedure, theefficacy of the treatment achieved, and the like. These compositionscontain an effective amount of the phototherapeutic agent along withconventional pharmaceutical carriers and excipients appropriate for thetype of administration contemplated. These compositions may also includestabilizing agents and skin penetration enhancing agents and also maycontain pharmaceutically acceptable buffers, emulsifiers, surfactants,and, optionally, electrolytes such as sodium chloride.

Formulations for enteral administration may vary widely as is well knownin the art. In general, such formulations are liquids, which include aneffective amount of the composition in an aqueous solution orsuspension. Such enteral compositions may optionally include buffers,surfactants, emulsifiers, thixotropic agents, and the like. Compositionsfor oral administration may also contain flavoring agents and otheringredients for enhancing their organoleptic qualities. A topicalapplication can be formulated as a liquid solution, water/oil emulsion,or suspension of particles, depending on the particular nature of theagent and the type of tissue to be targeted. If the azo compound iswater soluble, for instance, a solution in water may be applied to orinto the target tissue. The delivery of the azo compounds into andthrough the skin may be enhanced by using well known methods and agentssuch as transdermal permeation enhancers, for example, “azone”,N-alkylcyclic amides, dimethylsulfoxide, long-chained aliphatic acids(C10), etc. If the azo compound is not water soluble, it may bedissolved in a biocompatible oil (soybean oil, fish oil, vitamin E,linseed oil, vegetable oil, glyceride esters, long-chained fatty esters,etc.) and emulsified with surface-active compounds (vegetable or animalphospholipids; lecithin; long-chained fatty salts and alcohols;Pluronics: polyethylene glycol esters and ethers; etc.) in water to makea topical cream, suspension, water/oil emulsion, water/oilmicroemulsion, or liposomal suspension to be delivered or applied to thetarget region. In the case of liposomes, the azo compound may beattached to or be contained in the lamellar material.

The dose of the photosensitizer may vary from about 0.1 mg/kg bodyweight to about 500 mg/kg body weight. In one embodiment, the dose is inthe range of about 0.5 to 2 mg/kg body weight. As one example, forcompositions administered parenterally, a sterile aqueous solution orsuspension of the photosensitizer may be present in a concentrationranging from about 1 nM to about 0.5 M, typically in a concentrationfrom about 1 μM to about 10 mM.

In general, a formulated azo compound is administered at a dose or in aconcentration which is effective, upon exposure to light, to partiallyor completely inactivate a target tissue within a biological medium. Thebiological medium is exposed for a period of time to light of awavelength that is effective to activate the dye which produces type Idestruction in the target tissue. The concentration of the azo compoundat the target tissue is the outcome of either passive or active uptakeprocesses in the tissue. An example of passive uptake would be where theazo compound is attached or is contained within a particulate carrier.If the carrier is of an appropriate size, in the range of about 100 nmto about 1000 nm, it will “leak” into the perfusion boundary of vasculartumors. An example of active uptake would be where a receptor basedattachment binds a particular receptor that is expressed on the targettissue. The effective concentration of the azo compound is thusdependent on the nature of the formulation, method of delivery, targettissue, activation method and toxicity of the azo to the surroundingnormal tissue.

The following example illustrates a specific embodiment of the inventionpertaining to the preparation and properties of a typical bioconjugatederived from bombesin, a bioactive peptide, and a phototherapeuticmolecule, the azocoumarin derivative 11b as shown in FIG. 4.

EXAMPLE

Synthesis of azocoumarin-bombesin (7-14) conjugate

The peptide is prepared by fluorenylmethoxycarbonyl (Fmoc) solid phasepeptide synthesis strategy with a commercial peptide synthesizer fromApplied Biosystems (Model 432A SYNERGY Peptide Synthesizer). The firstpeptide cartridge containes Wang resin pre-loaded with an amide resin on25-μmole scale. The amino acid cartridges are placed on the peptidesynthesizer and the product is synthesized from the C- to the N-terminalposition.

Coupling of the Fmoc-protected amino acids (75 μmol) to the resin-boundfree terminal amine (25 μmol) is carried out with2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU, 75 μmol)/N-hydroxybenzotriazole (HOBt, 75 μmol). Each Fmocprotecting group on the solid support is removed with 20% piperidine indimethylformamide before a subsequent amino acid is coupled to it. Thelast cartridge contains the azo compound 11b as shown in FIG. 4, whichis coupled to the peptide automatically, thus avoiding the need forpost-synthetic manipulations.

After the synthesis is completed, the product is cleaved from the solidsupport with a cleavage mixture containing trifluoroacetic acid(85%):water (5%):phenol (5%):thioanisole (5%) for six hours. Thepeptide-azide conjugate is precipitated with t-butyl methyl ether andlyophilized in water/acetonitrile (2:3) mixture. The conjugate ispurified by high performance liquid chromatography (HPLC) and analyzedwith liquid chromatography/mass spectroscopy (LC/MS).

It should be understood that the embodiments of the present inventionshown and described in the specification are only specific embodimentsof the inventors who are skilled in the art and are not limiting in anyway. Therefore, various changes, modifications or alterations to thoseembodiments may be made or resorted to without departing from the spiritof the invention and the scope of the following claims. For example, thecompounds containing polycyclic aromatic chromophores can also be usedfor optical diagnostic imaging purposes.

What is claimed is:
 1. A compound of formula

wherein Q is a single bond or —CR¹R²; R¹ and R² are independentlyselected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10aryl, C1-C10 alkoxyalkyl, C1-C10 polyhydroxyalkyl, —(CH₂)_(a)CO₂H, and—(CH₂)_(b)NR³R⁴; R³ and R⁴ are independently selected from the groupconsisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, C1-C10polyhydroxyalkyl, and —(CH₂)_(a)CO₂H; R⁵, R⁶, and R⁷ are independentlyselected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10aryl, hydroxyl, —SO₃H, C1-C10 alkoxyl, C1-C10 polyhydroxyalkyl, C1-C10polyalkoxyalkyl, —(CH₂)_(a)CO₂H, and —(CH₂)_(b)NR³R⁴; X is selected fromthe group consisting of —CR⁸R⁹, —O—, —NR³, —S—, and —C═O; Y is selectedfrom the group consisting of —CR¹⁰R¹¹, —O—, —NR³, —S—, and —C═O; Z isselected from the group consisting of —CR¹²R¹³, —O—, —NR³, —S—, and—C═O; R⁸ to R¹³ are independently selected from the group consisting ofhydrogen, C1-C10 alkyl, C5-C10 aryl, C1-C10 alkoxyalkyl, C1-C10polyhydroxyalkyl, —(CH₂)_(a)CO₂H, and —(CH₂)_(b)NR³R⁴; R⁵-R⁶, R⁶-R⁷,R⁸-R¹⁰, or R¹⁰-R¹² together optionally form a six-membered ring; E iseither a hydrogen atom or is selected from the group comprisingantibodies, peptides, peptidomimetics, carbohydrates, glycomimetics,drugs, hormones, or nucleic acids; L is the linker unit —(CH₂)_(c)—, aand b independently range from 0 to 10, and c ranges from 1 to
 10. 2.The compound of claim 1, wherein Q is —CR¹R²; R¹ and R² areindependently selected from the group consisting of hydrogen and C1-C10alkyl; R⁵, R⁶, and R⁷ are independently selected from the groupconsisting of hydrogen, hydroxyl, —SO₃H, and —(CH₂)_(a)CO₂H; X is —C═O;Y is —CR¹⁰R¹¹; Z is —CR¹²R¹³; R¹⁰ to R¹³ are independently selected fromthe group consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, and—(CH₂)_(a)CO₂H; E is selected from the group consisting of somatostatinreceptor binding molecules, heat-sensitive bacterioendotoxin (ST)receptor binding molecules, neurotensin receptor binding molecules,bombesin receptor binding molecules, cholecystekinen (CCK) receptorbinding molecule, steroid receptor binding molecules, and carbohydratereceptor binding molecules; and a independently ranges from 0 to
 6. 3.The compound of claim 1, wherein Q is —CR¹R²; R¹ and R² areindependently selected from the group consisting of hydrogen and C1-C10alkyl; R⁵, R⁶, and R⁷ are independently selected from the groupconsisting of hydrogen, hydroxyl, —SO₃H, and —(CH₂)_(a)CO₂H; X is—CR⁸R⁹; Y is —C═O; Z is selected from the group consisting of —CR¹²R¹³,—O—, and —NR³; R⁸, R⁹, R¹² and R¹³ are independently selected from thegroup consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, and—(CH₂)₈CO₂H; E is selected from the group consisting of somatostatinreceptor binding molecules, ST receptor binding molecules, neurotensinreceptor binding molecules, bombesin receptor binding molecules, CCKreceptor binding molecule, steroid receptor binding molecules, andcarbohydrate receptor binding molecules; and a independently ranges from0 to
 6. 4. The compound of claim 1, wherein Q is —CR¹R²; R¹ and R² areindependently selected from the group consisting of hydrogen and C1-C10alkyl; R⁵, R⁶, and R⁷ are independently selected from the groupconsisting of hydrogen, hydroxyl, —SO₃H, and —(CH₂)_(a)CO₂H; X is—CR⁸R⁹; Y is selected from the group consisting of —CR¹⁰R¹¹, —O—, and—NR³; Z is —C=O; R⁸ to R¹¹ are independently selected from the groupconsisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, and —(CH₂)_(a)CO₂H; Eis selected from the group consisting of somatostatin receptor bindingmolecules, ST receptor binding molecules, neurotensin receptor bindingmolecules, bombesin receptor binding molecules, CCK receptor bindingmolecule, steroid receptor binding molecules, and carbohydrate receptorbinding molecules; and a independently ranges from 0 to
 6. 5. Thecompound of claim 1, wherein Q is —CR¹R²; R¹ and R² are independentlyselected from the group consisting of hydrogen and C1-C10 alkyl; R⁵, R⁶,and R⁷ are independently selected from the group consisting of hydrogen,hydroxyl, —SO₃H, and —(CH₂)_(a)CO₂H; X is —CR⁸R⁹; Y is —CR¹⁰R¹¹; Z is—CR¹²R¹³; R⁸ and R¹⁰ together form a benzene ring; R⁹ and R¹¹ areradicals that form carbon-carbon bond; R¹² and R¹³ are independentlyselected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10aryl, and —(CH₂)_(a)CO₂H; E is selected from the group consisting ofsomatostatin receptor binding molecules, ST receptor binding molecules,neurotensin receptor binding molecules, bombesin receptor bindingmolecules, CCK receptor binding molecule, steroid receptor bindingmolecules, and carbohydrate receptor binding molecules; and aindependently ranges from 0 to
 6. 6. The compound of claim 1, wherein Qis —CR¹R²; R¹ and R² are independently selected from the groupconsisting of hydrogen and C1-C10 alkyl; R⁵, R⁶, and R⁷ areindependently selected from the group consisting of hydrogen, hydroxyl,—SO₃H, and —(CH₂)_(a)CO₂H; X is —CR⁸R⁹; Y is —CR¹⁰R¹¹; Z is —O—; R⁸ andR¹⁰ together form a benzene ring; R⁹ and R¹¹ are radicals that formcarbon-carbon bond, R¹² and R¹³ are independently selected from thegroup consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, and—(CH₂)_(a)CO₂H; E is selected from the group consisting of somatostatinreceptor binding molecules, ST receptor binding molecules, neurotensinreceptor binding molecules, bombesin receptor binding molecules, CCKreceptor binding molecule, steroid receptor binding molecules, andcarbohydrate receptor binding molecules; and a independently ranges from0 to
 6. 7. The compound of claim 1, wherein Q is —CR¹R²; R¹ and R² areindependently selected from the group consisting of hydrogen and C1-C10alkyl; R⁵, R⁶, and R⁷ are independently selected from the groupconsisting of hydrogen, hydroxyl, —SO₃H, and —(CH₂)_(a)CO₂H; X is—CR⁸R⁹; Y is —CR¹⁰R¹¹; Z is —NR³; R⁸ and R¹⁰ together form a benzenering; R⁹ and R¹¹ are radicals that form carbon-carbon bond; R¹² and R¹³are independently selected from the group consisting of hydrogen, C1-C10alkyl, C5-C10 aryl, and —(CH₂)_(a)CO₂H; E is selected from the groupconsisting of somatostatin receptor binding molecules, ST receptorbinding molecules, neurotensin receptor binding molecules, bombesinreceptor binding molecules, CCK receptor binding molecule, steroidreceptor binding molecules, and carbohydrate receptor binding molecules;and a independently ranges from 0 to
 6. 8. The compound of claim 1,wherein Q is —CR¹R²; R¹ and R² are independently selected from the groupconsisting of hydrogen and C1-C10 alkyl; R⁵, R⁶, and R⁷ areindependently selected from the group consisting of hydrogen, hydroxyl,—SO₃H, and —(CH₂)_(a)CO₂H; X is —CR⁸R⁹; Y is —CR¹⁰R¹¹; Z is —C═O; R⁸ andR¹⁰ together form a benzene ring; R⁹ and R¹¹ are radicals that formcarbon-carbon bond; R¹² and R¹³ are independently selected from thegroup consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, and—(CH₂)_(a)CO₂H; E is selected from the group consisting of somatostatinreceptor binding molecules, ST receptor binding molecules, neurotensinreceptor binding molecules, bombesin receptor binding molecules, CCKreceptor binding molecule, steroid receptor binding molecules, andcarbohydrate receptor binding molecules; and a independently ranges from0 to 6.